The present invention is of polynucleotide sequences encoding alkaline-α-galactosidases and methods of using same.
The α-galactosidase enzyme (α-D-galactoside galactohydrolase) catalyzes the hydrolysis of the terminal linked α-galactose moiety from galactose-containing oligosaccharides. These include, for example, the naturally occurring disaccharide melibiose (6-O-α-D-galactopyranosyl-D-glucose), the trisaccharide raffinose (O-α-D-galactopyranosyl-(1-6)-O-α-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside) and the tetrasaccharide stachyose (O-α-D-galactopyranosyl-(1-6)-O-α-D-galactopyranosyl-(1-6)-O-α-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside).
α-galactosidases are classified into two families of the glycosyl hydrolase enzymes, with eukaryotic enzymes falling primarily into family 27 and prokaryotic enzymes primarily into family 36 [Henrissat A. et al. (1995) Biochem. J. 311:351-352 and Henrissat A. et al. (1991) Biochem. J. 280:309-316]. The α-galactosidases are also classified as acid or alkaline, depending on the pH of optimal activity. Most of the eukaryotic α-galactosidases studied to date are acidic α-galactosidases, with a broad pH optima in the acidic range [Keller F. and Pharr D. M. In: Zamski, E. and Schaffer, A. A. (eds.) Photoassimilate Partitioning in Plants and Crops: Source-Sink Relationships, ch. 7, pp. 168-171, 1996, Marcel Dekker Publ., N.Y.].
α-galactosidases have potential use in a variety of applications. [Margolles-Clark et al. (1996) Eur. J. Biochemistry, 240:104-111 and U.S. Pat. Nos. 5,633,130, 6,197,566 and 5,919,690 each of which is herein incorporated by reference in its entirety]; They may hydrolyze α-galactose residues from polymeric galactomannans, such as in guar gum, where modification of guar gum galactomannan with α-galactosidase has been used to improve the gelling properties of the polysaccharide [Bulpin, P. V., et al (1990) Carbohydrate Polymers 12:155-168]; α-galactosidase can hydrolyze raffinose from beet sugar syrup, which can be used to facilitate the sugar crystallization from molasses [Suzuki et al(1969) Agr. Biol. Chem., 33:501-513].
Additionally, α-galactosidases can also be used to hydrolyze stachyose and raffinose in soybean milk, thereby reducing or eliminating the undesirable digestive side effects which are associated with soybean milk [Thananunkal et al. (1976) Jour. Food Science, 41:173-175) and to remove the terminal α-galactose residue from other glycans, such as the erythrocyte surface antigen conferring blood group B specificity which has potential medical use in transfusion therapy by converting blood group type B to universal donor type O [Harpaz et al.(1975) Archives of Biochemistry and Biophysics, 170:676-683; and Zhu et al. (1996) Archives of Biochemistry and Biophysics, 327:324-329].
However, the use of acidic α-galactosidases in biotechnological and industrial applications is limited by the pH needed for activity. For instance, the use of an acidic form of α-galactosidase to remove the galactose-containing oligosaccharides, which include raffinose and stachyose, from soybean milk is difficult, as the pH of soybean milk, which is 6.2-6.4, is well above the optimum pH range of the Mortariella vinacea enzyme, which is 4.0-4.5, as shown using the natural substrate melibiose. Lowering the pH of the soybean milk solution to conform to the acidic pH optimum of this enzyme causes the soybean proteins to precipitate thus imparting a sour taste to the milk [Thanaunkul et al. (1976) Jour. Food Science, 41:173-175, 1976].
Likewise, use of α-galactosidase with an acidic pH optimum for the removal of raffinose from beet sugar faces a similar problem. The pH of the beet molasses has to be lowered to 5.2 with sulfuric acid in order for the Mortariella vinacea enzyme to function [Suzuki et al. (1969) Agr. Biol. Chem., 33:501-513].
The standard procedure for seroconversion requires the transfer of centrifuged erythrocytes to an acidic buffer in order for the acidic enzyme to function [Goldstein et al. (1982) Science 215:168-170, 1982]. However, lowering the pH for optimal activity of the coffee bean α-galactosidase causes the cells to be less stable thereby leading to cell lysis. Thus, seroconversion is carried out at pH 5.6, which reflects a compromise between red cell viability and optimal α-galactosidase activity [Zhu et al. (1996) Archives of Biochemistry and Biophysics, 327:324-329].
An additional limitation facing industrial application of α-galactosidases is that the product of the reaction, namely galactose, frequently inhibits their activity. For example, the reported alkaline α-galactosidase from Cucurbita pepo leaves is strongly inhibited by α-galactose [Geaudreault, P. R. and Webb, J. A. (1983) Plant Physiol., 71, 662-668].
Despite their importance in various commercial applications, only a few examples of eukaryotic alkaline α-galactosidases have been reported.
A plant alkaline-α-galactosidase with pH optima of 7-7.5 was initially discovered in young leaves of Cucurbita pepo [Gaudreault and Webb (1982) Plant Sci. Lett. 24:281-288, (1983) Plant Physiol. 71:662-668 and (1986) Plant Science 45:71-75]. This alkaline form has been reported to be stachyose specific, with only low affinity for raffinose and melibiose. Thus, this previously reported alkaline α-galactosidase could be described as having activity at alkaline pH but with only a narrow spectrum of substrates. Further characterization showed that α-D-galactose, the product of the enzymatic reaction, is a strong inhibitor of the enzyme's activity [Gaudreault and Webb (1983) Plant Physiol. 71:662-668], similar to many of the acid α-galactosidases.
It has been suggested that the alkaline-α-galactosidase from young leaves of cucurbit plays an important physiological role in phloem unloading and catabolism of transported stachyose in the young cucurbit leaf tissue, as it is the initial enzyme in the metabolic pathway of stachyose and raffinose catabolism. Likewise, it has been suggested that the enzyme may play an important role in the carbohydrate partitioning in melon plants, and may have possible functions for phloem unloading in fruits of muskmelon [Gaudreault P R and Webb J A (1986) Plant Science 45:71-75].
Recently, α-galactosidase activity at alkaline pH has been observed in other cucurbit tissue, such as cucumber fruit pedicels, young squash fruit and young melon fruit [Pharr and Hubbard (1994) Encyclopedia of Agricultural Science vol. 3 pp. 25-37]. All these observations suggest that stachyose degradation by α-galactosidase take place within pedicels of fruit of Cucumis sativus, especially in the regions where the pedicel joins the fruit.
A major reservation to the above described alkaline activity stems from the fact that all of these studies were carried out using the non-specific artificial substrate, p-nitrophenyl α-galactopyranoside (pNPG), which indicates α-galactosidase activity but does not reflect either the physiological role of the particular enzyme form, or, more importantly, the substrate specificity of the particular enzyme. Since it is well established that the artificial substrate pNPG often indicates a higher pH optimum for α-galactosidase activity than that observed with the natural substrates [Courtois and Petek (1966) Methods in Enzymology 8:565-571], the prior art does not teach the exact nature of the in-vivo activity of the above described alkaline α-galactosidase enzyme.
Alkaline α-galactosidase activity has been recently reported in plant families other than the Cucurbit family [Bachmann et al. Plant Physiology 105:1335-1345, 1994]. Though these are only very preliminary results accompanied by limited biochemical data, it indicates the possibility that alkaline α-galactosidases, including novel enzymes not previously characterized, may function throughout the plant kingdom.
While reducing the present invention to practice the present inventors have cloned two novel alkaline-α-galactosidase genes, which represent a previously unidentified glycosyl hydrolase family of alkaline-α-galactosidase which is similar to the yet uncharacterized seed imbibition protein (SIP) family.
Further characterization of these genes and their protein products has revealed that the enzymes of the present inventions have optimal activity at neutral to alkaline pH conditions (7-9) together with broad substrate specificity, as opposed to previously reported alkaline-α-galactosidases
Thus, the present invention provides novel polynucleotide sequences encoding alkaline-α-galactosidases and methods of using same for producing recombinant proteins, for determining the germination potential of seeds as well as other applications.